Na-Cl co-transporter (NCC) is a 125-kDa 12-transmembrane Na+-dependent cation-chloride co-transporter that is primarily expressed in kidney distal convoluted tubules to resorb the filtered load of NaCl in the kidney. In mice, deficiency of NCC causes Gitelman syndrome, an inherited hypokalemic alkalosis with significant hypocalciuria and hypomagnesemia, which are secondary to the deficit in NaCl resorption. Interleukin 18 (IL18) is an inflammatory cytokine that promotes inflammatory cell cytokine/chemokine (e.g., IFN-?, IL6, MCP-1) production. In atherosclerotic lesions, increased levels of IL18 and its cognate receptor IL18r are detected in macrophages, smooth muscle cells (SMCs), and endothelial cells (ECs). Serums IL18 levels are elevated in patients with CAD, correlate with CAD severity score, are associated with cardiovascular risk factors, and predict future CAD events. In experimental atherosclerosis, overexpression or intraperitoneal administration of IL18 increases atherosclerotic lesion formation and enhances vulnerable plaque phenotypes via an IFN-?- dependent pathway, while inactivation or genetic deficiency of IL18 slows lesion progression. Our preliminary studies show that IL18 binds to NCC and mediates downstream protein tyrosine phosphorylation in mouse heart ECs. Stimulation of macrophages, T cells, and aortic SMCs and ECs with inflammatory cytokines (e.g., IL18, TNF-?, IL1) induces NCC expression. In both humans and mice, normal aortas express negligible NCC and IL18r, but SMCs, ECs, and macrophages in atherosclerotic lesions express high amounts of both NCC and IL18r. In atherosclerosis-prone apolipoprotein E-deficient (Apoe-/-) mice, the absence of either IL18r or NCC does not slow atherosclerosis development significantly. But combined deficiency of IL18r and NCC significantly reduces aortic arch macrophage content and lesion areas, and thoracic-abdominal aorta lipid deposition, along with reduced serum cytokines IFN-? and IL6. From cultured macrophages, the combined absence of NCC and IL18r impairs IL18-mediated cell signaling, and significantly reduces IL18 cell-surface binding and consequent inflammatory cytokine and chemokine production. These observations lead to our central hypothesis that NCC and IL18r are alternative IL18-binding molecules on inflammatory cells and cardiovascular cells, and that they mediate IL18 activities cooperatively in atherogenesis and possibly in other inflammatory diseases. We propose three aims: 1). to examine whether inhibition, activation, or genetic deficiency of NCC affects atherogenesis; whether NCC activities on inflammatory cells (e.g. macrophages) are essential to atherogenesis; and age-dependent; 2). To examine whether NCC acts like other kinase receptor, NCC activities in salt uptake and IL18 signaling interplay with each other, IL18 has different affinity for NCC and IL18r, and NCC is specific for IL18; and 3). To examine whether NCC mediates IL18 activities in T cells and vascular SMCs and ECs.
Na-Cl co-transporter (NCC) is a 12-transmembrane cation-chloride co-transporter, expressed in the kidney distal convoluted tubules. We discovered that this ion channel co-transporter mediates IL18 signaling and cytokine production in ECs, macrophages, and NCC-transfected COS-7 cells. Induced NCC expression in macrophages, T cells, and vascular SMCs and ECs after inflammatory cytokine stimulation, increased NCC expression in atherosclerotic lesions, and impaired atherosclerosis in IL18-deficient Apoe-/- mice, but not in cognate IL18 receptor IL18r-deficient Apoe-/- mice, leading to our central hypothesis that this cell-surface ion channel protein is a novel alternative IL18-binding protein and cooperates with IL18r in mediating IL18 inflammation during atherogenesis, and possibly in other IL18-associated inflammatory diseases.
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